The Masson’s trichrome staining procedure

Masson’s trichrome staining assay was conducted to evaluate the inflammatory response of the muscle layer using a trichrome stain kit (ab150686, Abcam, US). Sections are deparaffinized and hydrated in distilled water. The slide is placed in 56–64 °C preheated Bouin’s fluid (60 min) followed by a cooling period (10 min). The slide is rinsed until the section is clear by water. The sections are stained with working Weigert’s iron hematoxylin for 5 min. After rinsing the slide, Biebrich scarlet/acid fuchsin solution is applied to the slide for 15 min. After rinsing the slide, the section is differentiated in phosphomolybdic/phosphotungstic acid solution until the collagen is not red. Aniline blue solution is applied to the slide for 5–10 min. After rinsing the slide, acetic acid solution (1%) is applied to the slide for 3–5 min. Samples are dehydrated in two changes of 95% alcohol.